CN103861146A - Bacterial cellulose biological patch and manufacturing method thereof - Google Patents
Bacterial cellulose biological patch and manufacturing method thereof Download PDFInfo
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- CN103861146A CN103861146A CN201410089918.8A CN201410089918A CN103861146A CN 103861146 A CN103861146 A CN 103861146A CN 201410089918 A CN201410089918 A CN 201410089918A CN 103861146 A CN103861146 A CN 103861146A
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Abstract
The invention relates to a bacterial cellulose biological patch and a manufacturing method thereof. The biological patch comprises a bacterial cellulose film, namely BC film, wherein the polymerization degree of the bacterial cellulose ranges from 2000 to 20000, the crystal form of the cellulose crystal is I type, the crystallization index is 50-95%, and in cell parameters, a is equal to 0.815nm, b is equal to 1.025nm, c is equal to 0.832nm, and beta is equal to 85 degrees. The bacterial cellulose biological patch has excellent mechanical property, better biological fitness, better anti-adhesion property and antimicrobial property in the enterocoelia environment, and proper area can be conveniently obtained.
Description
Technical field
The present invention relates to a kind of Bacterial cellulose product, more particularly, the present invention relates to a kind of medical Bacterial cellulose and manufacture method thereof.
Background technology
Some surgical operation of abdomen inner chamber need to adopt the balanced good sticking patch of physiology and mechanical property to repair fascia or tendon, typical case and common surgical diseases as this in abdominal hernia, and various countries crowd's sickness rate is always higher, is about 12%.Particularly incisional hernia, because meeting causes local chronic pain, abdominal distention discomfort, and the serious complication such as strangulated intestinal obstruction, intestinal perforation, tend to the Long-term Effect and patient's quality of life, even can threaten patient's life, adopt clinically sticking patch to carry out repairing hernia.The fairly large use sticking patch of China has also had more than ten years, and the demand of sticking patch is also increasing gradually.
At present, the sticking patch of domestic listing is by comprising that American-European 12 companies that wait provide, and more than 20 plant totally.These sticking patch are mainly synthetic materials, can cause in various degree some complication:
Polypropylene sticking patch can produce serious adhesion, digestive tract with intraperitoneal tissue and block even intestinal fistula.Fastness and anti-infection ability after expanded polytetrafluoroethylsealing sticking patch is repaired are poor, could infection control once there is to infect the necessary patching material of removing.The absorbable materials such as polyglycolic acid net, polylactic-co-glycolic acid net have good anti-infection ability, can promote the propagation of collagen.The compound hernia patching material that polypropylene combines with expanded polytetrafluoroethylsealing and with absorbable material can make to organize well to grow into, and can prevent again the adhesion with internal organs.But these macromolecule sticking patch only play barrier effect after inserting in abdomen, can not organically combine by body tissue, meanwhile, as foreign body, after inserting, can cause local organization inflammation and infection.
Therefore, further research and development can realize the bioactive materials of endogenous tissue regeneration, improve the medical level that state's entocele is repaired, improve therapeutic effect, reduce domestic medical expense, and even invent a kind of artificial material as fascia or tendon sample, cure hernia completely, become an important directions in hernia patching material field.
As desirable biological activity sticking patch, the condition that hernia patching material must be satisfied:
(1) tensile strength is greater than the maximum intra-abdominal pressure 16N/cm that human body bears, and corresponding strain is greater than 14%.
(2) the smaller the better with local inflammation scope and the antixenic degree of persistence of abdominal wall tissue and internal organs; Compared with macromolecule sticking patch tissue repairing process, can regenerate defective tissue is repaired by endogenous tissue.This endogenous tissue regenerative process is closer to the natural repair process of vital tissues, and the tissue after reparation does not have excessive scar tissue, does not have polymer foreign body to retain in vivo and the long-term chronic inflammatory process that occurs.
(3) microbial resistance: patching material performance and structure should more meet psychological need, and quality is lighter, and complication should be still less, are applicable to Intraabdominal transplanting, can optimize cicatrization process, adapts to contaminated or has a wound of contaminated risk.
(4) good adhesion inhibiting properties.
(5) size of sticking patch will enough adapt to defect area area.
Summary of the invention
For the problems referred to above of conventional art, the invention provides a kind of Bacterial cellulose biological sticking patch and manufacture method thereof, its tool has the following advantages: can in abdominal cavity environment, have good mechanical property, better biocompatibility, better adhesion inhibiting properties and microbial resistance, and can make expediently suitable area.
For this reason, one of technical solution of the present invention is a kind of Bacterial cellulose biological sticking patch, this biological sticking patch is that BC film forms by bacteria cellulose film, and the degree of polymerization of described Bacterial cellulose is in 2000～20000 scopes, the crystal formation of this cellulose crystallization is I type, and crystallization index is 50～95%, a=0.815nm in cell parameter, b=1.025nm, c=0.832nm, β=85 degree.
Bacterial cellulose of the present invention (Bacterial cellulose, BC) made by the acetobacter xylinum in acetobacter (Acetobacter xylinum) metabolite, this Pseudomonas has the highest cellulose production capacity, therefore can obtain easily the sticking patch goods of suitable area.
Bacterial cellulose of the present invention is identical in chemical composition with other source fiber element, all to have glucopyranose monomer (β-D-Glucose) by β-1,4-glycosidic bond connect and the one that forms without branch, macromole branch polymer, but in microstructure, have an obvious difference.Because the special microstructure of Bacterial cellulose BC of the present invention obtains following a lot of excellent performance:
Bacterial cellulose of the present invention is made up of unique filamentary fibers, and fibre diameter is between 3～4nm, and each filamentary fibers bandwidth can reach 30～100nm, and thickness can reach 3～8nm, and structure is very fine and close; Bacterial cellulose of the present invention very pure (reaching 99%), containing hemicellulose, lignin, pectin and other cell wall components; Bacterial cellulose also has high-crystallinity and the degree of polymerization simultaneously.This special structure makes Bacterial cellulose have extremely excellent mechanical performance, and its elastic modelling quantity is up to 1.5 × 10
10pa, suitable with metallic aluminium, much larger than current known organic polymer.And the tensile strength of single bacteria cellulose fibre silk and steel and Ke Weila fiber (Kevlar, the product of E.I.Du Pont Company, for the preparation of flak jackets) are suitable.Therefore it has the splendid anti-tear ability of drawing and shape maintains ability, be convenient to make various shapes, for example be applicable to the repairing of human body different parts tendon: after repairings for comprising that the hernia of various constitutionales and recurrent indirect inguinal hernia, straight hernia and femoral hernia is sickly repaired, after esophageal carcinoma excision, pleura reparation, skull reparation, breast-enlarging operation and the reparation of female pelvic cavity internal organs prolapsus, and be used as cardiovascular sticking patch, endomeninx sticking patch.
Simultaneously the fibrous reticular structure of Bacterial cellulose of the present invention makes it have outstanding retentiveness, can be in conjunction with than the moisture content of large 60～700 times of self dry weight.And the Bacterial cellulose that is both extracellular matrix has similar structure to collagen, all has the diameter of 100nm, Bacterial cellulose can replace collagen to use in bioengineered tissue material under many circumstances.Its nanofibrous structures can irritation cell contact abduction mechanism, make that cell can along the jut of material surface or fiber be orientated and moves.From distant view, this magical cellulose being synthesized by natural biology of the present invention will be made long-range contribution to the development of bioengineered tissue material.
As measured data below proves, Bacterial cellulose BC of the present invention, tool has the following advantages:
(1) BC has the three-dimensional network nanofibrous structures of the regeneration that is beneficial to newborn abdominal wall tissue.
(2) moisture content of BC is not less than 94%.
(3) tensile strength of BC material in the time that draw speed is 80mm/min is 27.87N/cm, and elastic strain is 54.8%, is greater than the marginal value of internal milieu needs, meets mechanical property and requirement that sticking patch should have.
The adhesion inhibiting properties in vitro study of BC and conventional P P shows: BC patching material of the present invention is having the following advantages aspect the adhesion that prevents peritoneum and abdominal viscera:
(5) there is the biological characteristics that promotes Peritoneal Mesothelial Cells growth, be conducive to form biological barrier;
(6) there is the effect that suppresses fibroblast proliferation and differentiation;
(7) suppress hematoblastic gathering, anti-hemostasis-coagulation;
(8) suppress fibrinogenic deposition.
Therefore Bacterial cellulose of the present invention is applicable to replacing traditional sticking patch, repairing for intraperitoneal surgical operation very much.
In order further to optimize microstructure, to improve Bacterial cellulose mechanics and physiological property and effect, cellulose products of the present invention also comprises following structural behaviour improvement:
In I crystal formation, the ratio of I α kenel is 50 – 85%.
Described bacteria cellulose film dry film density is 1.08 – 2.35g cm
-3; And after its water holding moisture content moistening, the weight rate of Bacterial cellulose is 0.14-15%, moisture content ratio is 85 – 99.86%(wt).
After moisture content moistening, the thickness of described cellulose membrane is: 0.1～1.0mm, and the diameter of single fiber element is 5～20nm, the diameter of cellulose bundle is in 10～80nm scope.
Maximum strain ε m:45.7～54.8% of described bacteria cellulose film after moisture content moistening, maximum fracture strength σ m:8.24～11.940Mpa, the maximum tensile strength Sm:20.73～27.865N/cm.
After moisture content moistening, described BC film is 0.86～1.72 to albuminous adsorbance with its ratio to fibrinogenic adsorbance; Described BC film absorption albumin and fibrinogenic time of equilibrium adsorption are 1-2 hour.
Correspondingly, another technical solution of the present invention is a kind of manufacture method of Bacterial cellulose biological sticking patch described above, its be select α-distortion Gammaproteobacteria (Alphaproteobacteria) subordinate's of Proteobacteria (Proteobacteria) subordinate Rhodospirillales be Rhodospirillales subordinate's acetobacter section be Acetobacteraceae subordinate's Acetobacter sp. be Acetobacter subordinate's acetobacter xylinum Acetobacter xylinum as bacterial strain, described manufacture method comprises the steps:
A) shaken cultivation: will normally activate in the cooled culture medium of bacterial strain access sterilizing shaken cultivation 24 – 36 hours at 30 DEG C ± 2 DEG C;
B) disperse bacterial strain: after shaken cultivation, culture medium is placed in shaking table taking rotating speed as 150～200rpm,, fully disperse bacterial strain;
C) leave standstill and cultivate: after scatter operation, culture medium constant temperature at 30 DEG C ± 2 DEG C leaves standstill to be cultivated 3～4 weeks;
D) cross leaching film: leave standstill after cultivation, then from culture medium, take out BC produced film, obtain BC filter caudacoria by stainless steel cloth membrane filtration;
E) clean masking: BC filter caudacoria and be immersed in 1 – 4%(wt) in NaOH solution, in 100 DEG C of boiling water of 90 –, heat 1 – 3 hours, remove tropina and stick to the residual media on cellulose membrane, neutralize with dilute hydrochloric acid, then repeatedly rinse to neutrality with deionized water, make described BC film finished product.
The manufacture method of Bacterial cellulose of the present invention preferably has the bacterial strain of excellence aspect very high cellulose production capacity,, crystallization process synthetic at cellulose and structural property, adopt for epigamic cultural method step, impel bacterial strain metabolism to produce mechanics and the balanced good cellulose of biological property.
In order more to give full play to and to optimize the production advantage of the inventive method in cellulose microstructure, further improve adaptability and the benefit of the inventive method, manufacture method of the present invention also comprises following improvement:
Also comprise the steps A0 before described steps A) bacterial strain activation: by freezing bacterial strain, activate in cultivating base filling activation nutritional solution, then carry out follow-up cultivation; In described step e, after cleaning operation, also comprise drying process.
The culture medium nutritional solution of described steps A comprises following component: (NH
2)
2sO
4: 2～4g/L, MgSO
4: 0.1～0.6g/L, KH
2pO
4: 1～3g/L, NaAc:1～2g/L, sucrose: 10～40g/L, Sucus Cocois: 400～600g/L; Sterilizing 15min at above-mentioned culture medium nutritional solution control pH=4.2,100 DEG C.
The activation nutritional solution of described steps A 0 comprises following component: sucrose 2%, peptone 0.5%, yeast extract 0.5%, sodium hydrogen phosphate 0.27%, citric acid 0.115%; Sterilizing 15min at above-mentioned activation nutritional solution control pH=6.0,115 DEG C.
Below in conjunction with drawings and the specific embodiments, the present invention will be further described.
Brief description of the drawings
Fig. 1 is the transmission electron microscope photo schematic diagram of BC embodiment of the present invention.
Fig. 2 is the transmission electron microscope photo schematic diagram of conventional P P.
Fig. 3 is the mechanical test performance curve schematic diagram of BC embodiment of the present invention.
Fig. 4 is the mechanical test performance curve schematic diagram of conventional P P.
Fig. 5 is BC embodiment of the present invention and the upper relative rate of increase RGR of the mesothelial cell value comparison diagram of conventional P P.
Fig. 6 is BC embodiment of the present invention and the upper relative rate of increase RGR of the L929 cell value comparison diagram of conventional P P.
Fig. 7 is the upper albumin/fibrin adsorbance ratio comparison diagram of BC embodiment of the present invention and conventional P P.
Fig. 8 is BC embodiment of the present invention and conventional P P comparison diagram of external dynamic blood coagulation time.
Fig. 9 is 8 weeks adhesion situation photographic view of BC embodiment repairing operation of the present invention.
Figure 10 is 8 weeks adhesion situation photographic view of conventional P P repairing operation.
Figure 11 is 8 weeks histopathology microphotograms of BC embodiment repairing operation of the present invention.
Figure 12 is 8 weeks histopathology microphotograms of conventional P P repairing operation.
Figure 13 is 8 weeks mesothelial cell's microphotograms of BC embodiment repairing operation of the present invention.
Figure 14 is 8 weeks mesothelial cell's microphotograms of conventional P P repairing operation.
Figure 15 is the inner microphotograms of BC embodiment repairing operation of the present invention 8 pericyte.
Figure 16 is the inner microphotograms of conventional P P repairing operation 8 pericyte.
Detailed description of the invention
The Rhodospirillales Rhodospirillales subordinate's of α-distortion Gammaproteobacteria (Alphaproteobacteria) of selection Proteobacteria (Proteobacteria) subordinate the Acetobacteraceae subordinate's of acetobacter section Acetobacter sp. Acetobacter subordinate's acetobacter xylinum Acetobacter xylinum is as bacterial strain, and described manufacture method comprises the steps:
A0) bacterial strain activation: by freezing bacterial strain, activate in cultivating base filling activation nutritional solution, activation nutritional solution comprises following component: sucrose 2%, peptone 0.5%, yeast extract 0.5%, sodium hydrogen phosphate 0.27%, citric acid 0.115%; Sterilizing 15min at above-mentioned activation nutritional solution control pH=6.0,115 DEG C.Then carry out follow-up cultivation;
A) shaken cultivation: will normally activate in the cooled culture medium of bacterial strain access sterilizing shaken cultivation 24 – 36 hours at 30 DEG C ± 2 DEG C; Culture medium nutritional solution comprises following component: (NH
2)
2sO
4: 2～4g/L, MgSO
4: 0.1～0.6g/L, KH
2pO
4: 1～3g/L, NaAc:1～2g/L, sucrose: 10～40g/L, Sucus Cocois: 400～600g/L; Sterilizing 15min at above-mentioned culture medium nutritional solution control pH=4.2,100 DEG C.
B) disperse bacterial strain: after shaken cultivation, culture medium is placed in shaking table taking rotating speed as 150～200rpm,, fully disperse bacterial strain;
C) leave standstill and cultivate: after scatter operation, culture medium constant temperature at 30 DEG C ± 2 DEG C leaves standstill cultivates 3～4
D) cross leaching film: leave standstill after cultivation, then from culture medium, take out BC produced film, obtain BC filter caudacoria by stainless steel cloth membrane filtration;
E) clean masking: BC filter caudacoria and be immersed in 1 – 4%(wt) in NaOH solution, in 100 DEG C of boiling water of 90 –, heat 1 – 3 hours, remove tropina and stick to the residual media on cellulose membrane, neutralize with dilute hydrochloric acid, then repeatedly rinse to neutrality with deionized water, carry out again drying process, make described BC film finished product.
The Bacterial cellulose making through aforesaid operations step is the biological sticking patch that BC film forms, record through physical and chemical testing: the degree of polymerization of described Bacterial cellulose is in 2000～20000 scopes, crystallization index is that 50～95%(XRD analyzes), the crystal formation of cellulose crystallization is I type, a=0.815nm in cell parameter, b=1.025nm, c=0.832nm, β=85 degree.In I crystal formation, the ratio of I α kenel is that 50 – 85%(NMR analyze).
Described bacteria cellulose film dry film density is 1.08 – 2.35g cm
-3; And after its water holding moistening, the weight rate of Bacterial cellulose is 0.14-15%, moisture content ratio is 85 – 99.86%(wt).
After moisture content moistening, the thickness of described cellulose membrane is: 0.1～1.0mm, and the diameter of single fiber element is 5～20nm, the diameter of cellulose bundle is in 10～80nm scope (Electronic Speculum test).
Maximum strain ε m:45.7～54.8% of described bacteria cellulose film after moisture content moistening, maximum fracture strength σ m:8.24～11.940Mpa, the maximum tensile strength Sm:20.73～27.865N/cm(mechanical test).
After moisture content moistening, described BC film is 0.86～1.72 to albuminous adsorbance with its ratio to fibrinogenic adsorbance; Described BC film absorption albumin and fibrinogenic time of equilibrium adsorption are 1-2 hour (In vitro culture test).
For relatively BC of the present invention is as the effect of biological sticking patch, we adopt the sticking patch of PP (polypropylene) material to be used as comparison.Below the serial contrast test of embodiment of the present invention BC sticking patch and conventional polypropylene PP sticking patch:
As Fig. 1,2, shown in be respectively the transmission electron microscope photo schematic diagram of BC embodiment of the present invention and conventional P P.Conventional P P polypropylene sticking patch is formed by polypropylene monofilament fibrage, has good intensity and rigidity.Its voidage reaches 85%, and density is 18.9g/m
2.
(1) water absorption experiment:
Because PP's is moist little, be the material substantially not absorbing water, therefore its moisture content is not measured.This experiment has only been done water suction experiment to BC:
In the surface plate of constant weight in advance, take Bacterial cellulose, in 100 DEG C of baking ovens, dry to constant weight;
Moisture content (%)=(front sample quality (g) of rear sample quality (g) × 100%/oven dry of sample quality (g)-oven dry before drying
Table 1
Moisture content (%)=(12.1236-0.6283) × 100%/12.1236=94.82%
(2) mechanical property experiment:
As Fig. 3,4, shown in be respectively the mechanical test performance curve schematic diagram of BC embodiment of the present invention and conventional P P.
2 kinds of materialogy performances are from test curve and following table 2: no matter the percentage elongation of BC is all lower than the percentage elongation of PP under which draw speed.Be increased to from 10mm/min the process of 200mm/min at draw speed Pulling speed, breaking strain when 80mm/min is the highest, BC's is 54.8%, PP's is 109.6%, the maximum strain ε m(54.8% of BC), fracture strength σ m(11.940MPa), tensile strength sm(27.865N/cm) although be all less than the PP sticking patch having gone on the market, but the marginal value (16N/cm) that is all greater than internal milieu needs, meets requirement used.
BC under wetting state has a small amount of moisture to extrude when by clamp and in drawing process.The drawing process of BC and PP all meets thin slice plastic deformation law, and the nearly middle position width of sample graticule narrows gradually, until final fracture can not be replied.
Table 2
Brief summary:
(1) BC has the three-dimensional network nanofibrous structures of the regeneration that is beneficial to newborn abdominal wall tissue.
(2) moisture content of BC is 94.82%.
(3) tensile strength of BC material in the time that draw speed is 80mm/min is 27.87N/cm, and elastic strain is 54.8%, is greater than the marginal value of internal milieu needs, meets requirement used.There is the mechanical property that sticking patch should have.
(3) the external adhesion inhibiting properties experiment of BC and PP:
As Fig. 5, be depicted as the upper relative rate of increase RGR(relative of the mesothelial cell growth rate of BC embodiment of the present invention and two kinds of different experiments materials of conventional P P (materials)) comparison diagram of value.
Mesothelial cell and BC, PP cultivate altogether 2d (my god) time, the relative rate of increase of cell is all lower than the relative rate of increase of negative (Negative) cellular control unit, especially the value of BC group is only 51.76%, can find out that mesothelial cell is slower in the propagation on BC surface.And because PP macroscopic view mesh is larger, cell based instinct adheres to growing multiplication through PP lacing in culture plate bottom, be not subject to the impact on lacing surface, therefore, the relative rate of increase of cell of PP group is higher is 82.49%.And along with the prolongation of incubation time, the relative rate of increase of cell when BC cultivates 4d altogether with PP all increases, and is respectively 77.26% and 90.22%, and the relative rate of increase increase rate of the cell on BC surface is higher than PP group.Therefore, can say that to a certain extent BC can promote that mesothelial cell breeds.
Mesothelial cell and material cultivate altogether 4d (my god) after, cellular control unit growth conditions is good, and the cell quantity on BC surface is few compared with matched group, PP group cell attaches the growth of culture plate bottom substantially completely, substantially be not subject to the impact on lacing surface, overall cell quantity is more than BC group cell quantity, close to cellular control unit quantity.And under scanning electron microscope, the mesothelial cell on BC surface is in good condition, ovalize or circle, cell surface has a large amount of intensive microvilluss, and the highdensity fine hair of cell peripheral forms highdensity haloing in background.
As Fig. 6, be depicted as the relative rate of increase RGR of l cell L929 value comparison diagram on two kinds of different experiments materials of BC embodiment of the present invention and conventional P P.
After cultivating altogether with BC and PP, the RGR value of L929 cell is all less than feminine gender (Negative) matched group, in PP group, RGR value and negative control group approach, the RGR value of BC group is minimum, illustrate that BC has hindered the propagation of L929, and along with the prolongation of time, BC and PP group cell RGR value all reduce, and have suppressed the propagation of L929.Illustrate that BC can suppress the propagation of L929.
The RGR value of BC and PP is all less than negative control group, and in PP group, RGR value and negative control group approach, and the RGR value of BC is minimum, illustrates that BC has hindered the propagation of L929, and along with the prolongation of time, BC and PP have all hindered the propagation of L929, especially more serious with BC obstruction.Illustrate that BC can suppress the propagation of L929.
Above-mentioned Fig. 5 experimental results show that: the mesothelial cell's that BC material surface attaches quantity increases along with the prolongation of incubation time, mesothelial cell is had to the effect that promotes growth; Above-mentioned Fig. 6 experimental results show that: the fibroblast of BC material surface reduces with the prolongation of incubation time, fibroblast is had to the effect that suppresses growth.
Thereby illustrate: BC patching material of the present invention has adhesion inhibiting properties, has following characteristics:
(1) there is the biological characteristics that promotes Peritoneal Mesothelial Cells growth, be conducive to form biological barrier effect;
(2) there is the effect that suppresses fibroblast proliferation and differentiation, there is the effect of preventing tissue adhesive.
As Fig. 7, being depicted as Absorption time is BC embodiment of the present invention and the above comparison diagram of albumin/fibrinous adsorbance ratio R (A/F) of conventional P P under the continuous adsorption time.
Because patching material surface adsorption albumin can suppress platelet adhesion reaction and thrombosis, and adsorbing fiber proteinogen is easy to make platelet adhesion reaction material surface, and then causes thrombocyte degeneration to be assembled, and secondary blood coagulation forms.
Experiment adopts bovine serum albumin (Albumin from bovine serum is called for short BSA) and Fibrinogen (Fibrin is called for short FN) to test the adhesion inhibiting properties of bi-material at the ratio R (A/F) of the adsorbance of material surface.The value of R (A/F) on Fig. 7 is higher, represents that material is just stronger to the inhibition of hematoblastic adhesion and gathering, and preventing adhesiving effect is just better.
Numerical value on Fig. 7 can show that through calculating BC sticking patch of the present invention and conventional P P sticking patch bi-material surface are to BSA(bovine serum albumin) and FN(Fibrinogen) the correction data of ratio R (A/F) of adsorbance as following table 3:
BSA and FN adsorbance ratio R (A/F) contrast on table 3 BC of the present invention and conventional P P sticking patch
| Time(min) | 10 | 30 | 60 | 120 | 360 |
| BC | 1.556 | 1.595 | 1.761 | 1.717 | 1.672 |
| PP | 0.361 | 0.579 | 0.659 | 0.653 | 0.682 |
Therefore: generally, the R on BC surface (A/F) is worth higher than PP surface.
More than experimental results show that:
Patching material of the present invention has adhesion inhibiting properties, has following characteristics:
(3) can suppress fibrinogenic deposition;
(4) can suppress hematoblastic gathering, anti-hemostasis-coagulation.
As Fig. 8, be depicted as BC embodiment of the present invention and the conventional P P comparison diagram of external dynamic blood coagulation time of absorbance OD reflection.Experiment can be demonstrate,proved, and the dynamic blood coagulation time of BC sticking patch, than the dynamic blood coagulation time lengthening of PP sticking patch, illustrates that the anticoagulation function of BC improves, and blood compatibility is better, can stop the generation of adhesion.The result of this test is consistent compared with high and fibrinogenic adsorbance compared with the result of low and difficult generation blood coagulation with albumin adsorbance in protein adsorption test.
the adhesion inhibiting properties experiment brief summary that BC and PP are external:
(1) mesothelial cell's that BC material surface attaches quantity increases along with the prolongation of incubation time, mesothelial cell is had to the effect that promotes growth; And the fibroblast on surface reduces with the prolongation of incubation time, fibroblast is had to the effect that suppresses growth.
(2) BC material reaches balance to bovine serum albumin and the fibrinogenic 2h of being adsorbed on left and right, and the ratio R (A/F) of the adsorbance of two kinds of albumen is 0.86～1.72, is greater than polyacrylicly 0.65, can suppress platelet adhesion and thrombosis.
(3) BC material has good anticoagulant property.
(4) the animal abdominal wall defect repairing of BC and PP test:
Method: get 5 SD male rat (rat; A strain of rattus norregicus, claims again: Sprague-Dawley rat), rat weighs after body weight, after 10% chloral hydrate anesthesia success, the clinostatism of making even, fixing rat, conventional skin is prepared, sterilization, paving aseptic towel.Get paramedian incision, successively each layer of structure of open abdomen is to abdomen fascia; Excision rat midriff muscular tissue 1.5cm × 1.5cm, gives hemostasis by ligation, successively enters abdomen, and attention protects intestinal tube and other intraperitoneal are tolerant, retains abdominal wall surface skin and subcutaneous tissue simultaneously.Dispose into 2 kinds of patching material: 2cm × 2cm in middle stomach wall, select absorbable thread, two kinds of patching material four angle seams inserting are closed and are fixed on abdominal wall muscle tissue.Select absorbable thread layer-by-layer suture to close abdomen.Meanwhile, get 5 normal rats and raise simultaneously, as blank group.
Judgment criteria:
◇ is postoperative is placed in sub-cage rearing in the Animal House that temperature and humidity is suitable by experimental rat, observes the ordinary circumstance of animal.Put to death rat when week (w be English week) respectively at postoperative 2w, 4w, 8w in batches;
◇ observes sticking patch surface adhesion situation;
◇ scanning, the mesothelial cell's survival of transmissioning electric mirror checking surface and regeneration situation;
◇ histopathological examination sticking patch and surrounding tissue ankylose situation.
Overview: a little feed water inlet of experimental group rat after kposthesis, food-intake was recovered normally substantially in the 3rd day, and this group rat is all without infecting, and does not occur obvious post-operative complication, without dead.This experimental group rat body weight is changed to carry out statistical analysis known, with the comparison of blank group rat, the last fortnight body weight change has significant difference (P<0.05), difference that in to eight time-of-week, there are no significant the following (P>0.05).The initial body weight gain of BC group rat slowly mainly by rat to implanting slightly uncomfortable causing of foreign body in operation process.
As Fig. 9, be depicted as 8 weeks adhesion situation photographic view of BC embodiment repairing operation of the present invention.(in figure, dark tetragon represents size and the position of sticking patch, inside has the light rectilinear frame that "×" is intersected to represent adhesion area and position.）
BC organizes rat, and postoperative body temperature is normal, and feed water inlet is normal, all, without infecting, does not occur obvious post-operative complication.2w after kposthesis, 4w, 8w uses laparotomy exploration after putting to death rat, draws materials, visible defect repairing place abdominal wall structure ideal; After kposthesis, 8w draws materials, can observe the repaired organizational structure in rat abdominal-wall defect place, the BC surface coverage translucent fibrous tissue of growing, and in thin layer tissue, there is blood capillary growth, with surrounding tissue without obvious adhesion, observe abdominal viscera smooth surface simultaneously, have no obvious inflammation adhesion, without cicatrix and granulation tissue growth.
After excision abdominal wall muscle tissue, abdominal-wall defect model basically forms, and raises after January, and abdominal-wall defect size slightly expands.Postoperative have a concurrent ecchymosis of rat, but postoperative 3 weeks can absorb completely.After abdominal-wall defect animal model is successful, the less generation of intra-abdominal adhesions, the average adhesion ratio after 5 rat experiments is 3.275%.
As Figure 10, be depicted as 8 weeks adhesion situation photographic view of conventional P P repairing operation.(in figure, dark tetragon represents size and the position of sticking patch, inside has the light rectilinear frame that "×" is intersected to represent adhesion area and position.）
After same abdominal-wall defect repairing operation, rats eating inflow is normal, after this PP group rat repairing operation, has 2 death, and laparotomy inspection finds in rat body that intestinal tube and sticking patch adhesion densification cause that intestinal blocks and cause death.All the other rats are dissected and draw materials at the appointed time time, find that PP sticking patch surface surrounding tissue all has large area adhesion.2w after kposthesis, 4w, 8w draws materials respectively, and can observe the rat abdominal-wall defect PP of place material surface surrounding tissue all has large area adhesion, as shown in figure 10.
As Figure 11, be depicted as 8 weeks histopathology microphotograms of BC embodiment repairing operation of the present invention.
In figure, evenly the dark point of sparse distribution is mainly fibrocyte, and dense distribution darker regions is inflammatory cell, and it is hemocyte that light color is punctuated.
After postoperative 2w and 8w, BC surface is mainly mesothelial cell, adipose cell and fibrocyte composition.2w time skin cell layer is thinner, and there is inflammatory reaction subregion, has a little multinucleated giant cell and neutrophil infiltration, inflammation 2-3 level, 2 grades of fibrosiss.There is newborn blood capillary.When 8w, inflammatory reaction is most of to disappear, and mesothelial cell's layer thickens, and ordered state is good, and seriality is good, and cell and BC combine closely.
Therefore, the good biocompatibility of BC, can promote mesothelial cell's regeneration.
As Figure 12, be depicted as 8 weeks histopathology microphotograms of conventional P P repairing operation.
In figure, evenly the dark point of sparse distribution is mainly fibrocyte, and dense distribution darker regions is inflammatory cell, and it is hemocyte that light color is punctuated.
After same abdominal-wall defect repairing operation, after 2w and 8w, PP surface and surrounding tissue are mainly neutrophilic granulocyte, lymphocyte and multinucleated giant cell composition, only have a small amount of mesothelial cell to form, and inflammation rank is higher, and inflammation 2-3 level has a large amount of necrocytosiss, abscess.When 8w, inflammation disappears not yet, still taking neutrophilic granulocyte and lymphocyte as main, and there is into fibrous tissue and form, have no typical mesothelial cell's arrangement form, have a small amount of adipose cell, form one deck fibrous connective tissue with adipose cell, belong to well differentiated fiber adhesion.Therefore, PP material is implanted the abdominal cavity damaged meeting of direct repairing and is caused serious inflammation foreign material repulsion reaction, stimulates around and becomes fibrous tissue growth, and have a large amount of multinucleated giant cells and neutrophilic granulocyte, and antigen-reactive is obvious.
Figure 13 is 8 weeks mesothelial cell SEM electron microscope photographing figure of BC embodiment repairing operation of the present invention.
While can be observed postoperative 2w under scanning electron microscope, BC surface mesothelial cell is more sparse, orientation is poor, and surface has more secretions to adhere to, and subregion is exposed, 4w is that mesothelial cell's growth conditions is better, surface secretions reduces, but still more sparse, subregion is exposed, when 8w, mesothelium growth is fine and close, iuntercellular connects intensive, and orientation is better, during having collagen fiber to distribute simultaneously.
As Figure 14, be depicted as 8 weeks mesothelial cell SEM electron microscope photographing figure of conventional P P repairing operation.
Under scanning electron microscope, can be observed in the 8w time of PP implantation, all do not find typical mesothelial cell's new life, only have the poor mesothelium growth of a small amount of state in subregion, structure is also more loose, when 8w, has fibrous tissue to form, the scar tissue that collagen forms.
Figure 15 is the inner TEM electron microscope photographing figure of BC embodiment repairing operation of the present invention 8 pericyte.
In figure, ater is hemocyte.Under transmission electron microscope, observe cell surface and have the microvillus that more or less in a large number, have abundant endoplasmic reticulum and mitochondrion in cytoplasm, meanwhile, cell peripheral has blood capillary growth (Figure 18).
As Figure 16, be depicted as the inner TEM electron microscope photographing figure of conventional P P repairing operation 8 pericyte.
Under transmission electron microscope, observe PP and show that a small amount of mesothelial cell's state is poor, the microvillus on surface is less, nucleus enlargement.
(5) abdominal-wall defect is repaired the surperficial adhesive confrontation ratio of BC and PP:
Sticking patch surface adhesion area and adhesion degree are calculated:
Respectively 3 groups, every group each 5 rats are carried out to abdominal-wall defect repairing operation with BC and PP sticking patch, surperficial adhesion character sampling is carried out in grouping in postoperative 2,4,8 weeks, adopt gridding method to calculate sticking patch surface adhesion area (0-100%), cut off the unnecessary adhesion organization in sticking patch surface, by after sticking patch nine deciles, nine points again of each little lattice, record adhesion area Calculating material surface adhesion ratio; The method that refers again to the scholars such as KA.LeBlanc is analyzed, and represents sticking patch surface adhesion degree with the digit score of 0-3.
The adhesion ratio of table 4 material surface
The adhesion ratio on BC and PP surface is as shown in table 4.Repairing in the 8w time, a group of BC surface adhesion ratio maximum is the 8w group of drawing materials, and is (8.50 ± 0.49) %, and a group of PP surface adhesion ratio maximum to be 4w draw materials group is (41.53 ± 0.35) %.Same time point, BC has compared significant difference with the adhesion ratio of PP group, has statistical significance (P<0.05).
The adhesion degree of table 5 material surface
Because the initial damaged area of stomach wall is limited, according to adhesion grading standard, the adhesion degree on BC and PP surface does not all occur 4 grades.BC surface does not have adhesion or slight adhesion substantially generally, and there is more serious adhesion on PP surface with organs and tissues.
Brief summary:
(1) 2w after kposthesis, 4w, when 8w, observes the repaired organizational structure in BC group rat abdominal-wall defect place, the BC surface coverage translucent fibrous tissue of growing, and have blood capillary to grow in thin layer tissue, and with surrounding tissue without obvious adhesion.Observe abdominal viscera smooth surface simultaneously, have no obvious inflammation adhesion, without cicatrix and granulation tissue growth.Histopathology and electron microscopic observation discovery, along with the prolongation of Implantation Time, mesothelial cell's quantity of BC surface regeneration increases, and iuntercellular connects intensive, and orientation is better, and state takes a turn for the better, and blood capillary is abundant.And PP group rat stomach wall mend is mainly neutrophilic granulocyte, lymphocyte and multinucleated giant cell composition, only there is a small amount of mesothelial cell to form, inflammation rank is higher, adhesion inflammation, a small amount of mesothelial cell's state is poor.
(2) within the repairing 8w time, BC surface adhesion ratio is (8.50 ± 0.49) % to the maximum, and adhesion degree is 0～1 grade; And PP surface adhesion ratio is (41.53 ± 0.35) % to the maximum.Adhesion degree is 2 grades.
(6) sum up:
(1) BC has the three-dimensional network nanofibrous structures that is conducive to newborn abdominal wall tissue regeneration.The tensile strength of BC material in the time that draw speed is 80mm/min is 27.87N/cm, and elastic strain is 54.8%, is greater than the marginal value of internal milieu needs, meets requirement used.There is the mechanical property that sticking patch should have.
(2) mesothelial cell's that BC material surface attaches quantity increases along with the prolongation of incubation time, mesothelial cell is had to the effect that promotes growth; And the fibroblast on surface reduces with the prolongation of incubation time, fibroblast is had to the effect that suppresses growth.
(3) BC reaches balance to bovine serum albumin and the fibrinogenic 2h of being adsorbed on left and right, and the ratio of the adsorbance of two kinds of albumen is 0.86～1.72, is greater than polyacrylicly 0.65, can suppress platelet adhesion and thrombosis.
(4) BC and surrounding tissue have good histocompatibility, and abdominal adhesions degree is lower, and can promote newborn mesothelial cell's regeneration.BC surface adhesion ratio is (8.50 ± 0.49) % to the maximum, and adhesion degree is 0-1 grade; And PP surface adhesion ratio is (41.53 ± 0.35) % to the maximum.Adhesion degree is 2 grades.
Therefore visible: BC of the present invention can make suitable area expediently, in the cavity environments such as human abdominal cavity, there is good mechanical property, better biocompatibility, better adhesion inhibiting properties and microbial resistance.
Claims (10)
1. a Bacterial cellulose biological sticking patch, this biological sticking patch is that BC film forms by bacteria cellulose film, it is characterized in that: the degree of polymerization of described Bacterial cellulose is in 2000～20000 scopes, the crystal formation of this cellulose crystallization is I type, crystallization index is 50～95%, a=0.815nm in cell parameter, b=1.025nm, c=0.832nm, β=85 degree.
2. Bacterial cellulose biological sticking patch as claimed in claim 1, is characterized in that: in I crystal formation, the ratio of I α kenel is 50 – 85%.
3. Bacterial cellulose biological sticking patch as claimed in claim 1 or 2, is characterized in that: described bacteria cellulose film dry film density is 1.08 – 2.35g cm
-3; And after its water holding moistening, the weight rate of Bacterial cellulose is 0.14-15%, moisture content ratio is 85 – 99.86%(wt).
4. Bacterial cellulose biological sticking patch as claimed in claim 1 or 2, is characterized in that: after moisture content moistening, the thickness of described cellulose membrane is: 0.1～1.0mm, and the diameter of single fiber element is 5～20nm, the diameter of cellulose bundle is in 10～80nm scope.
5. Bacterial cellulose biological sticking patch as claimed in claim 1 or 2, it is characterized in that: maximum strain ε m:45.7～54.8% of described bacteria cellulose film after moisture content moistening, maximum fracture strength σ m:8.24～11.940Mpa, the maximum tensile strength Sm:20.73～27.865N/cm.
6. Bacterial cellulose biological sticking patch as claimed in claim 1 or 2, is characterized in that: after moisture content moistening, described BC film is 0.86～1.72 to albuminous adsorbance with its ratio to fibrinogenic adsorbance; Described BC film absorption albumin and fibrinogenic time of equilibrium adsorption are 1-2 hour.
7. the manufacture method of a Bacterial cellulose biological sticking patch as described in one of claim 1-6, be select Rhodospirillales be Rhodospirillales subordinate's acetobacter section be Acetobacteraceae subordinate's Acetobacter sp. be Acetobacter subordinate's acetobacter xylinum Acetobacter xylinum as bacterial strain, described manufacture method comprises the steps:
A) shaken cultivation: will normally activate in the cooled culture medium of bacterial strain access sterilizing shaken cultivation 24 – 36 hours at 30 DEG C ± 2 DEG C;
B) disperse bacterial strain: after shaken cultivation, culture medium is placed in shaking table taking rotating speed as 150～200rpm,, fully disperse bacterial strain;
C) leave standstill and cultivate: after scatter operation, culture medium constant temperature at 30 DEG C ± 2 DEG C leaves standstill to be cultivated 3～4 weeks;
D) cross leaching film: leave standstill after cultivation, then from culture medium, take out BC produced film, obtain BC filter caudacoria by stainless steel cloth membrane filtration;
E) clean masking: BC filter caudacoria and be immersed in 1 – 4%(wt) in NaOH solution, in 100 DEG C of boiling water of 90 –, heat 1 – 3 hours, remove tropina and stick to the residual media on cellulose membrane, neutralize with dilute hydrochloric acid, then repeatedly rinse to neutrality with deionized water, make described BC film finished product.
8. the manufacture method of Bacterial cellulose biological sticking patch as claimed in claim 7, it is characterized in that: before described steps A, also comprise the steps A0) bacterial strain activation: by freezing bacterial strain, cultivate in base and activate filling activation nutritional solution, then carry out follow-up cultivation; In described step e, after cleaning operation, also comprise drying process.
9. the manufacture method of Bacterial cellulose biological sticking patch as claimed in claim 7, is characterized in that: the culture medium nutritional solution of described steps A comprises following component: (NH
2)
2sO
4: 2～4g/L, MgSO
4: 0.1～0.6g/L, KH
2pO
4: 1～3g/L, NaAc:1～2g/L, sucrose: 10～40g/L, Sucus Cocois: 400～600g/L; Sterilizing 15min at above-mentioned culture medium nutritional solution control pH=4.2,100 DEG C.
10. the manufacture method of Bacterial cellulose biological sticking patch as claimed in claim 8, is characterized in that: the activation nutritional solution of described steps A 0 comprises following component: sucrose 2%, peptone 0.5%, yeast extract 0.5%, sodium hydrogen phosphate 0.27%, citric acid 0.115%; Sterilizing 15min at above-mentioned activation nutritional solution control pH=6.0,115 DEG C.
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